Phenolic profiles and in vitro biochemical properties of Thai herb ingredients for chronic diseases prevention

Traditional remedies using natural ingredients have been handed down over generations, providing collective information for the development of modern therapeutics. These natural products have a long history of safe consumption with curative effects but lack of scientific-based evidence hinders the mass production of new remedies containing active ingredients with particular medicinal properties. This research investigated the phenolic profiles and biochemical properties of 29 herbal ingredients identified in two traditional Thai remedies, Prasachandaeng (PSCD) and Chantaharuethai (CHRT), and their effectiveness in combating chronic diseases. These two traditional remedies are used to reduce fever but some ingredients have been previously reported to possess other health-related properties. Statistical analysis by TOPSIS indicated that Biancaea sappan (L.) Tod. extract exhibited the highest overall closeness coefficient (C) score analyzed from all variables including total phenolic contents, antioxidant potentials, and enzyme inhibitions. Bouea macrophylla Griff. extract showed potential as an effective agent against diabetes through inhibition of two carbohydrate degrading enzymes, α-glucosidase and α-amylase, while Dischidia major (Vahl) Merr. showed strong properties as an anti-angiotensin-converting enzyme, leading to the control of hypertension. Dracaena cochinchinensis (Lour.) S.C. Chen effectively controlled the progression of Alzheimer’s disease through the inhibition of cholinergic and β-amyloid formation enzymes. These results can be used as preliminary data for the development of new remedies to prevent or treat particular chronic diseases.


Analysis of phenolic profile
Phenolic profiles of all herbs were investigated by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), while a well-established protocol along with LC-ESI-MS/MS parameters and validations was previously reported (with data in Supplementary Table 3) 15,16 .The dried extracts were redissolved in 62.5% (v/v) methanol and filtered through a 0.22 µm polytetrafluoroethylene (PTFE) syringe filter.The filtrate was loaded into a LC-ESI-MS/MS system consisting of a 2.1 mm × 100 mm, 2.6 μm Accucore RP-MS column, a Dionex Ultimate 3000 series ultrahigh-performance liquid chromatograph (UHPLC), a TSQ Quantis Triple Quadrupole mass spectrometer (MS), a diode array detector and a Chromeleon 7 chromatography data system (Thermo Fisher Scientific, Bremen, Germany).The gradient mobile phase used in this experiment is shown in Table 2.
Total phenolic contents (TPCs) of the extract were also determined utilizing a well-established protocol as previously reported without any modification 17 .Folin-Ciocalteu's phenol was used as a reagent and gallic acid concentration ranging 0-200 µg/mL was used as a standard.Absorbance at 765 nm was detected utilizing a Synergy™ HT 96-well UV-visible microplate reader and Gen 5 data analysis software (BioTek Instruments, Inc.,

Analysis of antioxidant activities
Antioxidant activities of the herbal extracts were determined using three assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric ion reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) assays as previously reported without any modification 17 .All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).Assay types, main reagents, and detection wavelengths are shown in Table 3.The reaction was detected using a microplate reader, and Trolox was used as a standard, with results expressed as µmol Trolox equivalent (TE)/g DW.

Analysis of enzyme inhibitory activities
The enzyme inhibitory activities of the herbal extracts were performed using AChE, BChE, BACE-1, ACE, α-amylase, and α-glucosidase assays using well-established protocols [18][19][20][21] , as summarized in Table 4.The AChE, BChE, α-amylase, and α-glucosidase inhibitory assays were kinetically measured using a Synergy™ HT UV-visible microplate reader and Gen 5 data analysis software (BioTek Instruments, Inc., Winooski, VT, USA), while the same instruments were used to determine the end-point inhibitory assays of BACE-1 and ACE.All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), with percentage of inhibition calculated using Eq. ( 2) as follows: where A is the initial velocity (V 0 ) of a reaction with an enzyme but without a herbal extract (control), a is the V 0 of the reaction without an enzyme and a herbal extract (control blank), B is the V 0 of a reaction with an enzyme and a herbal extract (sample), and b is the V 0 of a reaction with a herbal extract but without an enzyme (sample blank).

Statistical analysis
All experiments were performed in triplicate for three independent sets of samples (n = 3).Statistical analysis was determined utilizing one-way analysis of variance (ANOVA) with Duncan's multiple range test and significant differences set at p < 0.05.Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of TPCs, antioxidant potentials, and key enzyme inhibitory activities were determined using XLSTAT® (Addinsoft Inc., New York, NY, USA).The Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) comprehensive evaluation analysis was analyzed according to previous reports 22 .

Phenolic profiles and total phenolic contents
Phenolic profiles of all the herbal extracts were investigated utilizing LC-ESI-MS/MS with 24 authentic standards.Eight phenolic acids were detected in all extracts, as indicated in Table 5.Among the detected phenolic acids, most herbal extracts contained 3,4-dihydroxybenzoic acid (16 herbal extracts), followed by caffeic acid (10 herbal extracts), gallic acid and chlorogenic acid (8 herbal extracts), p-coumaric acid (4 herbal extracts), (2)  Overall, MF exhibited the highest phenolic acid contents using this analytical method, while no phenolic acids were detected in MET, KG, CA, GA, HT, and SE.Seven flavonoids were detected in the herbal extracts (Table 6).Most herbal extracts contained naringenin (17 herbal extracts), followed by rutin (9 herbal extracts), luteolin and apigenin (7 herbal extracts), isorhamnetin (6 herbal extracts), kaempferol (2 herbal extracts), and hesperidin (1 herbal extract).Naringenin was abundantly found in TC, while rutin was high in JS.PK contained the highest luteolin content, and apigenin was highly detected in AA.NN exhibited the highest contents of isorhamnetin and kaempferol, while hesperidin was only detected in CA.Overall, CA exhibited the highest flavonoid contents using this analytical method, while no phenolic acids were detected in CSP, MET, TH, GP, KG, LS, AD, and HT.
When combining phenolic acids and flavonoid contents, MF exhibited the highest phenolic content detected by the LC-ESI-MS/MS method, while no phenolics were detected in MET, KG, and HT.In comparison to TPCs analyzed by spectrophotometric methods using Folin-Ciocalteu's phenol as the reagent, TPCs of all herbal extracts ranged 26.31-728.50mg GAE/g extract with CSA exhibiting the highest TPC, while MET gave the lowest (Table 7).

Enzyme inhibitory activities
The inhibitory activities of the herbal extracts were assessed on the key enzymes that control the occurrence of some NCDs including type II diabetes (T2DM) (α-amylase and α-glucosidase), Alzheimer's disease (AChE, BChE, and BACE-1) and hypertension (ACE).Results indicated that the herbal extracts exhibited different degrees of inhibition against these enzymes using particular extract concentrations, as indicated in Table 8.
Carbohydrate digestive enzymes, α-amylase and α-glucosidase, are the therapeutic targets for diabetic drug design, and acarbose is a competitive inhibitor of these enzymes.Twenty-one herbal extracts exhibited α-amylase inhibitory activities ranging 0.85-92.66%using extract concentration of 0.25 mg/mL, while inhibitory activities www.nature.com/scientificreports/were not detected in 8 herbal extracts using the same extract concentration.Among the herbal extracts with α-amylase inhibitory activities, EAC and BM exhibited more than 90% inhibition, suggesting their high potentials as α-amylase inhibitors.These herbal extracts also exhibited higher degrees of inhibition against another carbohydrate degrading enzyme, α-glucosidase (ranging 4.63-98.52%inhibition using extract concentration of 0.06 mg/mL).Among the herbal extracts with α-glucosidase inhibitory activities, MF, NN, CL, UN, CSA, DL, EAC, BM, and DM exhibited more than 90% inhibition.Combining the data from these two carbohydrates hydrolyzing enzyme inhibitions, EAC and BM showed promise as antidiabetic agents.Two hypotheses of the mechanisms of Alzheimer's disease occurrence involve (i) termination of neurotransmitters by two cholinergic enzymes, AChE and BChE, and (ii) β-amyloid formation by BACE-1.Results indicated that at extract concentration of 0.20 mg/mL, all herbal extracts exhibited AChE inhibitory activities ranging 34.59-99.01%.Among these, MS, MF, MEF, NN, UN, CSA, DL, EAC, BM, DM, and SE exhibited AChE inhibitory activities at more than 90%.Likewise, their BChE inhibitory activities ranged 23.23-98.57%using extract concentration of 0.20 mg/mL.Among these, MS, MF, NN, UN, CSA, DL, EAC, PK, BM, and DM showed more than 90% inhibition, while no activity was detected in MET, TH, GP, and AD.Using the same extract concentration, BACE-1 inhibitory activities of 7.80-75.53%were observed in all herbal extracts, with the exception of EAC that had no inhibitory activity.CA exhibited the highest BACE-1 inhibitory activity but its AChE and BChE inhibitions were lower than 53%.When combining the inhibitory data from these three main enzymes that control Alzheimer's disease occurrence, DL and DM had more than 90% inhibition against AChE and BChE and more than 60% inhibition against BACE-1 as two potential herbal extracts for reducing the risk of Alzheimer's disease.www.nature.com/scientificreports/Inhibition of ACE, the main enzyme in reducing the risk of hypertension, was investigated using extract concentration of 1 mg/mL.Results indicated that all herbal extracts exhibited ACE inhibitory activities ranging 2.91-81.19%inhibition, with the exception of JS, CSP, TH, KG, AD, and HT with no ACE inhibitory activity detected.Among all the herbal extracts with detected ACE inhibitions, BM and DM exhibited more than 80% inhibition, suggesting their high potential as anti-ACE agents.

Principal component analysis
Principal component analysis (PCA) was used to further evaluate the data (Tables 7, 8).The PCA concept involves the reduction of large data sets into an interpretable figure while retaining as much of the original data as possible.To accomplish this, the mean values of TPCs, antioxidant activities (DPPP radical scavenging, FRAP, and ORAC activities), and enzyme inhibitory activities against key enzymes implicated in NCDs, such as α-amylase, α-glucosidase, AChE, BChE, BACE-1, and ACE were subjected to analysis.Data from Tables 7 and 8 were converted using PCA into a biplot, shown in Fig. 1, consisting of 2PCs including PC1 and PC2.The former covered 58.44% while the latter covered 17.65%, resulting in a total of 76.09% and indicating that the original data were represented and interpretable appropriately with minor error.PC1 contained α-amylase, α-glucosidase, AChE, BChE, and ACE inhibitory activities, while TPCs, antioxidant activities through DPPH radical scavenging, FRAP and ORAC assays, and BACE-1 inhibitory activities were positioned in PC2. Figure 1 illustrates the active variables (TPCs, antioxidant activities, and inhibitory activities against α-amylase, α-glucosidase, AChE, and BChE), Table 8.Inhibitory activities against α-amylase, α-glucosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), β-secretase (BACE-1) and angiotensin-converting enzyme (ACE) of 29 herbal extracts.All data are denoted as mean ± standard deviation (SD) of triplicate experiments (n = 3).List of abbreviation of samples are shown in Table 1.Different superscript letters indicate significantly different inhibitory activities of the same enzyme assay in different herbs (p < 0.05) using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test; AChE: acetylcholinesterase; BChE: butyrylcholinesterase; BACE-1: β-secretase; ACE: angiotensin-converting enzyme; ND: not detected; 1 final extract concentration = 0.25 mg/mL; 2 final extract concentration = 0.06 mg/mL; 3 final extract concentration = 0.20 mg/ mL; 4 final extract concentration = 1 mg/mL.www.nature.com/scientificreports/while BACE-1 inhibitory activities were clustered together, implying some correlation between these variables.More TPCs were related to high antioxidant activity, as evaluated by the three assays.BACE-1 inhibitory activities were located opposite to other variables; thus, herbal samples with high BACE-1 activities tended to have low activities compared to other variables.Figure 1 also shows that most of the active observations (plant samples; such as TH, CSP, AD, JS, KG, MET, and CA) were clustered opposite the active variables, suggesting that they had low values in those variables.Interestingly, CA, GA, DL, and SE with high BACE-1 inhibitory activities were not located close to its position, implying that BACE-1 activities were not unique for these plants, while UN, EAC, BM, and DM were clustered with the most active variables.Therefore, except for BACE-1 activities, these four plants were high in TPCs, antioxidant activities, and enzyme inhibitory activities against key enzymes implicated in NCDs such as amylase, glucosidase, AChE, BChE, and ACE.Further investigations on the phytochemical profiles are required to unravel the bioactive compounds in these plants.
The results of antioxidant and enzyme inhibitory activities from the 29 herbal samples were complicated and difficult to interpret; consequently, two independent statistical analyses were used to overcome this hurdle.First, TOPSIS analysis was applied for multiple criteria decision-making, using the mean data of 10 dependent variables (TPCs, antioxidant activities determined by DPPP radical scavenging, FRAP and ORAC assays, and inhibitory activities against α-amylase, α-glucosidase, AChE, BChE, BACE-1, and ACE).The weight ratio for each variable was set at 0.1 as they were equally important.The positive ideal solution of Euclidean distance (D+), negative ideal solution of Euclidean distance (D−), and the closeness coefficient (C) of all 29 plant samples were determined.Among all the herbal samples, CSA exhibited the highest C score (0.827) determined from antioxidant activities by both HAT (ORAC assay) and SET (DPPH radical scavenging and FRAP assay) mechanisms (Supplementary Table 5).This result indicated that the CSA herb sample exhibited the highest antioxidant potential.Likewise, the BM herb sample showed the highest potential to inhibit the carbohydrate degrading enzymes α-amylase and α-glucosidase, with the highest C score of 0.993 (Supplementary Table 6).For the enzymes involved in Alzheimer's disease (AChE, BChE, and BACE-1), DL exhibited the highest C score of 0.891 (Supplementary Table 7).Results in Table 8 showed that BM and DM enzymes involved in hypertension exhibited the highest ACE inhibitory activities.Overall, the plant with the highest C score was CSA (0.7588, Table 9), indicating that this sample had high TPC, antioxidant potential and enzyme-inhibiting activity.Conversely, AD (0.1337) harbored the lowest values of all tested variables.These statistical analyses demonstrate that among the 29 plant samples, CSA showed the highest potential in terms of TPC, antioxidant potential and enzyme-inhibiting activity.

Discussion
Traditional herbs have been widely used as effective remedies to treat many diseases, from trivial symptoms like common fever to more severe infections such as COVID-19.Some of these green medicines are popular among locals due to their confidence in safe consumption and successful treatments.However, scant scientific-based evidence is available to support their medicinal properties.The health-related properties of each ingredient of PSCD and CHRT might also show promise for developing new remedies as other potential NCD treatments.This study investigated the herbal ingredients in the Thai traditional remedies PSCD and CHRT for their potential on reducing the risk of NCDs.Among all the herbal samples, TOPSIS analyses indicated that (i) CSA exhibited the highest overall results for TPC, antioxidant potential, and enzyme inhibition; (ii) BM possessed high potential to inhibit diabetes through α-amylase and α-glucosidase inhibition; (iii) BM and DM exhibited the highest ACE inhibition, leading to the control of hypertension; and (iv) DL retarded Alzheimer's disease progression through cholinergic (AChE and BChE inhibitions) and β-amyloid formation (BACE-1 inhibition).
High TPCs in CSA gave high antioxidant potential in both HAT and SET mechanisms.A strong correlation between TPCs and antioxidant activities was also observed in rice, sacred lotus, and other plant extracts 12,16,23 , while water extract of CSA stem bark exhibited TPC of 682.67 mg GAE/g extract and DPPH radical scavenging activities with IC 50 of 9.47 µg/mL as the highest among 16 galactogogue medicinal plants in Northeastern Thailand 24 .This TPC value corresponded to our 70% (v/v) aqueous ethanolic extract of CSA with TPC of 728.50 mg GAE/g extract.Due to its high TPC, the CSA extract also exhibited overall high enzyme inhibition determined by TOPSIS analysis.Sappan Lignum, extracted with 80% (v/v) aqueous ethanol, exhibited α-glucosidase inhibitory activity with IC 50 of 6.3 µg/mL 25 , while an in vivo experiment indicated that CSA exhibited antidyslipidemic and antidiabetic activities in alloxan induced diabetic rat 26 .No previous studies have reported on the anti-Alzheimer's disease property of CSA, and this study is the first to report its anti-Alzheimer's disease potential through the inhibition of cholinesterases and BACE-1 activities.The phenolic profile analyzed by LC-ESI-MS/ MS indicated that CSA exhibited predominantly chlorogenic acid, followed by 3,4-dihydroxybenzoic acid and a trace of naringenin.CSA exhibited high TPC but its phenolic profile indicated moderate to low phenolic content, suggesting that CSA contained particular phenolics other than the common ones used as our LC-ESI-MS/MS standards.Previous research on phenolic profiles of CSA analyzed by liquid chromatography-quadrupole timeof-flight tandem mass spectrometry (LC-Q-TOF-MS/MS) found 4,7 dihydroxycoumarin as a major compound (24.47%area sum) with methyl 7-desoxypurpurogallin-7-carboxylate trimethyl ether, brazilein, and biochanin A (approximately (8% area sum) as the minor compounds 27 .These compounds added up to high TPC distribution in CSA.No previous report has been published on enzyme inhibitory activities of 4,7 dihydroxycoumarin, Table 9. TOPSIS data and rank calculated from the mean data of ten dependent variables from 29 plant samples determined using overall activities.List of abbreviation of samples are shown in Table 1.D+: positive ideal solution of Euclidean dis-tance; D−: negative ideal solution of Euclidean distance; C: the closeness coefficient.The weight ratio for each variable was equally set at 0.1.This compound also prevented hypertension in insulin resistant and type 2 diabetic rats 30 .An in-silico analysis suggested that dihydroxy coumarin interacted with the peroxisome proliferator-activated receptor (PPAR) but with lower binding affinity than orlistat (a competitive lipase inhibitor) 31 .These reports suggested that 4,7 dihydroxycoumarin might also exhibit these properties with different degrees of activity.Other bioactive compounds in CSA were previously reported for their health-related properties.A comprehensive pharmacologic investigation suggested that fisetin tetramethyl ether could be responsible for T2DM management through T2DM associated signaling pathways and target receptors including proliferator-activated receptor gamma (PPARG) 32 .
Other bioactive compounds in CSA including brazilin, sappanchalcone and protosappanins A-E, along with its methanolic extract, possessed vasorelaxant activities on rat aorta and the mesenteric artery 33 .BM strongly inhibited α-amylase and α-glucosidase, the key enzymes in T2DM control.Many plant parts including fruit, seed, stem, and leaves have been previously reported for BM 34 but no reports are available on its root.Thus, this is the first report on the phenolic profile and health-related properties of BM root.BM exhibited the second most abundant TPCs, with high levels of phenolic acids including gallic acid and 3,4-dihydioxybenzoic acid, moderate amounts of flavonoids including luteolin and naringenin detected by LC-ESI-MS/MS.As the most abundant phenolic detected in BM, gallic acid inhibited α-amylase with IC 50 ranging 4.35-13.69mM depending on the assay method 35 , while another study indicated that 3,4-dihydroxybenzoic acid exhibited a similar IC 50 value to gallic acid 36 .When compared to acarbose, a commercially available T2DM drug acting as an α-amylase and α-glucosidase competitive inhibitor, gallic acid and 3,4-dihydroxybenzoic acid were less effective, with IC 50 ranging 0.018-0.300mM against α-amylase 35 .The inhibitory effect of gallic acid was also observed in α-glucosidase assay, inhibiting α-glucosidase in a competitive manner with a 2.4-fold higher IC 50 value than acarbose (15.87 µM) 37 .However, 3,4-dihydroxybenzoic acid exhibited IC 50 of 3.64 mM against α-glucosidase 38 , suggesting that this phenolic inhibited α-glucosidase at a lower strength than gallic acid.
Two herbal extracts, BM and DM, strongly inhibited ACE, the key enzyme contributing to hypertension.Similar to BM, DM also exhibited high TPCs with gallic acid and 3,4-dihydroxybenzoic acid abundantly detected.A recent ethnopharmacological database only indicated traditional usage of DM 39 but its methanolic extract (unknown plant part) at a concentration of 1 mg/mL was unable to inhibit ACE 40 .At the same concentration, our aqueous ethanolic extract of DM exhibited 81.19% inhibition against ACE.As the most abundant phenolics, gallic acid exhibited IC 50 of 0.33 mM against ACE, while 3,4-dihydroxybenzoic acid exhibited < 50% inhibition at a concentration of 0.5 mM 41 .However, these phenolics exhibited lower ACE inhibitory strength than the commercially available ACE inhibitors captopril (IC 50 15.85-79.40nM), lisinopril (IC 50 5.62-31.6nM), and enalapril (IC 50 1.2-70.0µM) (IC 50 values varied depending on the tissues being tested) 42 .
Likewise, DL strongly inhibited cholinergic (AChE and BChE) and β-amyloid formation enzymes (BACE-1), thereby showing potential as an effective agent to control Alzheimer's disease.Interestingly, DL or Chinese Dragon's Blood has long been reported to exhibit neuroprotective effects through its bioactive compound, loureirin C 43 .Loureirin C was recently shown to improve cognitive impairment in Alzheimer's disease genetically modified mice via estrogen receptor α (ERα) 44 , while stemwood extract of DL prevented amyloid-β (Aβ) fibril formation and disassembled Aβ aggregation, leading to reduced Aβ-induced neuronal toxicity 7 .Several bioactive compounds were detected in DL including resveratrol, pterostilbene, loureirin B, and loureirin A that were able to prevent Aβ fibril-induced cell death; however, these compounds provided a weaker effect than crude DL extract 7 .Thus, the synergistic effect or other bioactive compounds might be responsible for this activity.Our LC-ESI-MS/ MS analysis indicated that DL exhibited high apigenin content with a moderate amount of naringenin, while no phenolic acid was detected in this herbal extract.Apigenin (25-100 µM) improved climbing ability in a transgenic Drosophila model of Alzheimer's disease, with similar results to the 0.1 mM donepezil treated group compared to unexposed AD flies 45 .The AD flies exposed to apigenin (50-100 µM) exhibited lower AChE activities and formation of Aβ aggregation than the unexposed AD flies 45 .A molecular docking investigation indicated AChE structural change due to the binding of apigenin at the peripheral binding site (flexible part that locates near the entrance of the AChE active site), indicating a noncompetitive type of inhibitor 46 .Thus, apigenin could be another bioactive compound in DL contributing to the retardment of Alzheimer's disease development.

Conclusion
Twenty-nine herb ingredients in two Thai traditional remedies, PSCD and CHRT, which are famous for their fever-lowering properties, were investigated for their phenolic profiles, antioxidant potentials, and inhibition of the key enzymes controlling the risk of NCDs.CSA exhibited the highest overall results on TPCs, antioxidant potentials, and all enzyme inhibitions.However, when considering each particular NCD, BM possessed high potential to control T2DM occurrence through inhibition of α-amylase and α-glucosidase, while BM and DM showed potential to ameliorate hypertension through ACE inhibition.DL controlled Alzheimer's disease progression through the cholinergic enzymes (AChE and BChE) and the β-amyloid forming enzyme (BACE-1).This preliminary information can be used to develop new herbal remedies or extracts for the prevention or treatment of NCDs.

Table 1 .
Scientific names, abbreviations, plant parts and voucher specimen of twenty-nine herbal ingredients used in this research.*Rotten wood.

Table 5 .
Phenolic acid profile of twenty-nine herbal extracts detected by a liquid chromatographyelectrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).All data are denoted as mean ± standard deviation (SD) of triplicate experiments (n = 3).List of abbreviation of samples are shown in Table1.Different lowercase letters indicate significantly different contents of the same phenolic detected in different samples and different capital letters indicate significantly different contents of different phenolics detected in the same sample at p < 0.05 using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test.ND, not detected.The antioxidant activities of all herbal extracts were investigated utilizing DPPH radical scavenging, FRAP, and ORAC assays.The first two assays follow single electron transfer (SET) mechanism of antioxidants, while the last follows the hydrogen atom transfer (HAT) mechanism.Results in Table7(with raw data in Supplementary Table4) indicated that all herbal extracts exhibited different degrees of antioxidant activities, ranging 0.12-2.35µmol TE/g extract for the DPPH radical scavenging assay, with DM exhibiting the highest activities and the lowest detected in GP and KG.All herbal extracts also exhibited FRAP activities ranging 156.23-5196.96µmol TE/g extract, with DM exhibiting the highest FRAP activity and MET, GP, and AD the lowest FRAP activity providers.The ORAC activities of all herbal extracts ranged 244.27-15,906.87 µmol TE/g extract.Similar to TPCs, CSA exhibited the highest ORAC activity, while MET gave the lowest.

Table 6 .
Flavonoid profile of twenty-nine herbal extracts detected by a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).All data are denoted as mean ± standard deviation (SD) of triplicate experiments (n = 3).List of abbreviation of samples are shown inTable 1. Different lowercase letters indicate significantly different contents of the same phenolic detected in different samples and different capital letters indicate significantly different contents of different phenolics detected in the same sample at p < 0.05 using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test.ND, not detected.

Table 7 .
Total phenolic contents (TPCs) and antioxidant activities determined by 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging, ferric ion reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays of twenty-nine herbal extracts.All data are denoted as mean ± standard deviation (SD) of triplicate experiments (n = 3).List of abbreviation of samples are shown in Table1.Different superscript letters indicate significantly different antioxidant activities determined by the same assay of different herbs (p < 0.05) using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test.GAE: gallic acid equivalent; TE: Trolox equivalent.